Signal transduction from cell-surface members of the interleukin-1 receptor/Toll-like receptor (IL-1R/TLR), tumor necrosis factor receptor (TNFR), and immunoglobulin (Ig) superfamilies coordinates the host defense against microbial infections. TNFR-associated factor 6 (TRAF6), a ubiquitin (Ub)-protein ligase (E3), underpins many of the TLR-dependent immune responses to pathogen-associated molecular patterns (PAMPs) expressed by bacteria and viruses. If left unchecked, these TRAF6-dependent responses can lead to excessive cytokine production, tissue injury, and other pathological consequences of inflammation. TRAF6 also mediates signaling from receptor activator of NF-?B (RANK) and CD40, TNFR family members involved in the differentiation and effector functions of osteoclasts and B lymphocytes, respectively. According to recent in vitro data, Lys-63 (K63)-linked ubiquitination of TRAF6 plays a key role in its mechanism of action by creating a docking site for cytosolic protein kinases that relay immunoreceptor signals to transcription factors NF-?B and AP-1. Despite these advances using in vitro models, the physiologic function and clinical relevance of TRAF6 ubiquitination remains untested. Similar to TRAF6, NF-?B essential modulator (NEMO) is subject to K63- linked ubiquitination. Prior experiments with transformed T cells suggested that this Ub modification to NEMO regulates antigen receptor (AgR) signaling to NF-:B. However, in vivo experiments reported here reveal impaired TLR rather than AgR signaling in "knock-in" mice harboring a point mutation that removes the Ub acceptor site of NEMO. This discovery underscores the need to test downstream consequences of Ub conjugation in a physiologic setting and establishes the feasibility of a complementary project to determine the in vivo significance of TRAF6 ubiquitination. Using a similar knock-in approach, mice will be engineered to harbor a mutation that selectively blocks K63-linked ubiquitination of TRAF6 but not its Ub-ligase activity (Aim 1). Subsequent phenotypic studies will focus on defects associated with TRAF6 deficiency that impinge on hematopoiesis and osteoclastogenesis (Aim 1), innate immunity versus pathologic inflammation (Aim 2), and adaptive immunity (Aim 3). Biochemical studies of immunoreceptor signaling to NF-?B and AP-1 will be conducted with primary cells affected by the germline mutation. Importantly, this analysis will define the function of K63-linked ubiquitination at the level of TRAF6 under conditions that ensure its correct spatial, temporal, and quantitative expression, which cannot be achieved via conventional transfection approaches. In vivo data accrued from the proposed project will advance knowledge about the functional workscope of TRAF6-Ub conjugates well beyond the current limitations of in vitro systems, a prerequisite for assessing the potential value of K63-linked polyubiquitination as a therapeutic target in inflammation-based disease. Public Health Relevance: Signal transmission within cells of the immune system coordinates the host defense against microbial pathogens and must be tightly regulated to avoid chronic inflammation. Recent in vitro experiments suggest that signal transmission involves the attachment of atypical ubiquitin chains to the intracellular protein TRAF6, which in turn stimulates the expression of host defense genes. New in vivo studies are proposed to investigate the physiologic function of this specific protein modification, its relevance to human health, and the potential for treating inflammation-based disease at the level of TRAF6 ubiquitination.